The synthesis of polypeptides by conventional methods in the solid phase, amino acid by amino acid, is limited by low yields when the polypeptides synthesized are large. Assembling two polypeptides by chemical ligation to produce a longer polypeptide is a known way of overcoming this limitation.
The complete synthesis of polypeptides is being used increasingly for preparing proteins with well-defined structures or bearing natural modifications, such as post-translational modifications, or non-natural modifications. The methods of chemical ligation provide an answer to this need, but they prove to be limited in their use and their industrial application.
In general, in these methods it is desirable for the bond between the polypeptides assembled by ligation to be native, i.e. to correspond to the natural structure of the polypeptides.
At present the main method for native ligation is that of Kent and Dawson, described for example in international applications WO 96/34878 and WO 98/28434. This method is based on a chemoselective reaction between a (C-terminal) thioester peptide and a cysteinyl-peptide. However, the main drawback of this method is production of the thioester peptides, which requires complex chemical methods. These methods cannot prevent competition between the reactions of the different thioesters, which inevitably leads to mixtures that may be difficult to separate, and therefore affecting the purity of the end product obtained, and to inevitable losses of yield.
An alternative method is the so-called Staudinger ligation, described in international applications WO 01/68565 and WO 01/87920. This comprises reaction of a phosphinothioester with an azide and hydrolysis of the combined reactants to form an amide bond. However, this method is difficult to apply on an industrial scale.
Another method, described in international application WO 2007/037812, is based on the reaction of an α-keto acid with an N-alkoxyamine in a decarboxylative condensation reaction. However, the keto acids are molecules that are difficult to manufacture and to incorporate in peptides. Thus, this third method is difficult to apply in peptide synthesis laboratories that do not possess means for carrying out complex organic syntheses.
The work by O. Melnyk et al., Org. Lett., 12(22), 5238-41 (2010) and application FR-2952058, as well as the work by Hou, W., Zhang, X., Li, F. and Liu, C. F. Peptidyl N,N-Bis(2-mercaptoethyl)-amides as Thioester Precursors for Native Chemical Ligation. Org. Lett. 13, 386-389 (2011), describe the native ligation of peptides by means of peptide-bis(sulphanylethyl)amino fragments. However, this method has never been used, to date, for the synthesis of peptides by assembling 3 or more fragments.
Application WO2011/058188 describes a purification technique consisting of introducing, at the end of solid-phase peptide synthesis, an N-terminal linkage provided with an azide function. Using a Staudinger-Bertozzi reaction or a cycloaddition (CuAAC, SPAAC), it is possible to graft the target peptide on a hydro-compatible resin functionalized beforehand with a phosphine or an alkyne. The peptide is finally released by cleavage of the linkage under mild conditions (base, nucleophilic, or photoirradiation) after washing the resin to remove the truncated peptides that had been unable to bind covalently with the resin. However, it is not envisaged at all in this document to use the grafting means for carrying out the synthesis of proteins by successive ligation of peptides.
The article by M. Villain et al., Chemistry and Biology 8 (2001) 673-679 describes a purification technique comprising a step in which the peptide comprising a cysteine or a threonine at the N-terminal end is grafted onto a resin, at the end of peptide synthesis, by reaction of the N-terminal residue with an aldehyde function, so as to form a thiazolidine or oxazolidine ring. The grafted peptide is then washed and then detached from the resin. However, it is not envisaged at all in this document to use the grafting means for carrying out the synthesis of proteins by successive ligation of peptides.
The documents U.S. Pat. No. 7,884,182 and Synthesis of Peptide-PNA-Peptide Conjugates by Semi-Solid-Phase Chemical Ligation Combined with Deactivation/Capture of Excess Reactants, Martijn C. de Koning. Eur. J. Org. Chem. 2004, 850-857, describe ligation of peptides using a solid support. The authors attach an H-Cys-A-SR peptide (R=alkyl) onto this support by the formation of a thiazolidine bond, then carry out a native ligation NCL between the supported thioester function and an H-Cys-B peptide. The H-Cys-A-SR peptides are difficult to synthesize. This method has a high risk of the occurrence of a polymerization or cyclization reaction during formation of the thiazolidine, since the two ends are reactive. Finally, this method does not allow more than one ligation to be carried out on the support.
Document FR 2 952 058 describes a method of ligation of peptides in the liquid phase, in the C-terminal to N-terminal direction. This method involves:                the use of a SEA peptide,        optionally the use of a SEAoff peptide, which is then converted to SEA.        
Document US 2002/0132975 describes a method of assembling peptides by sequential native ligation of peptide fragments in the solid phase. The synthesis can be performed from the N-terminal end to the C-terminal end or in the opposite direction. It is based on:                the use of peptides bearing an N-terminal cysteine and a C-terminal COS- group,        conversion of the -COS- function to -COSR thioester at the C-terminal end of the resin-supported peptide,        the use of the C-terminal thioester functionality of the resin-supported peptide for carrying out each ligation on the terminal cysteine of the next peptide fragment. However, the thioacid peptides (i.e. bearing the COS- function), such as those described as starting products in application US 2002/0132975, are difficult to prepare by synthesis, difficult to purify, and they are unstable. Moreover, the step of activation of the thioacid function to thioester is difficult especially when there are free cysteines in the peptide sequence, since there is a risk of alkylating the cysteines as well. Finally, the thioacid group, in contrast to the SEAoff group, is a reactive group that is not blocked. This is in particular illustrated in the work by Canne, L. E. et al., J. Am. Chem. Soc. 1999, 121, 8720-8727, in which the authors found significant formation of a cyclic by-product resulting from the residual reactivity of the thioacid group.        
Relative to the methods of the prior art, the method of the invention has the benefit of greater efficacy of conversion of the SEAoff function to thioester, the reaction conditions are milder and they do not lead to the formation of by-products. Moreover, SEAoff is a system that is blocked under the ligation conditions, in contrast to other reactive groups, and in particular the thioacid described in application US 2002/0132975. Finally, the SEAoff segments are synthesized easily in the solid phase by the Fmoc/tert-butyl strategy, which is not the case with the thioacid peptides.
The adaptation of methods making it possible to implement peptide syntheses by complete synthesis on an industrial scale is a need that requires finding methods that are simple, inexpensive, and produce quality products of high purity, and are acceptable in terms of industrial hygiene.
For the reasons stated above, it has become essential to find a method of complete synthesis that is convergent and capable of industrial application, making it possible to synthesize a peptide chain of the desired length and nature; in particular, a method involving assembly from the N-terminal to the C-terminal, which offers qualities of simplicity of execution and purity of the peptides or polypeptides obtained. In fact, assembly from the N-terminal to the C-terminal offers a considerable advantage, compared to the far more conventional opposite strategy of assembly from the C-terminal to the N-terminal, as in this case the method allows “self-purification” of the peptide fragments by removing the acetylated N-truncated peptides, major impurities in solid-phase peptide synthesis (SPPS).
The present invention makes it possible to overcome the difficulties associated with multiple ligations in solution. It makes it possible to synthesize very large proteins. It can easily be automated and extrapolated to the industrial scale.